L-type Ca2+ channels (LTCCs) play a fundamental role in brain neurons as mediators of diverse Ca2+ signaling events. LTCCs on neuronal somata play a unique and crucial role in regulating Ca2+-dependent gene expression. A salient feature of LTCCs is that their activity is regulated by clustering through cooperative gating of clustered channels. Their clustering also localizes them to specialized Ca2+ signaling microdomains within which they functionally couple to Ca2+-dependent proteins that transduce the impact of LTCC-mediated Ca2+ entry to specific Ca2+ signaling pathways. Through their canonical function as K+ conducting voltage-gated channels, somatic Kv2.1 channels play critical roles in the regulation of action potentials, with a subsequent impact on LTCC activity. The general consensus is that the functions of LTCCs and Kv2.1 channels in neurons are otherwise largely independent from one another. Our recent work challenges this view. We discovered a novel and unexpected nonconducting role for Kv2.1 in physically regulating the organization of neuronal LTCCs, enhancing their activity and impacting their localization in specific microdomains. These exciting new results lead to a novel model that in brain neurons, Kv2.1 plays dual roles, one as a canonical K+ channel shaping the intrinsic membrane properties of neurons, and the other a nonconducting physical role to cluster LTCCs to enhance their activity and localize them in Ca2+ signaling microdomains. The combination of the complementary backgrounds and skill sets of the Timmer and Santana labs allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the molecular mechanisms whereby Kv2.1 impacts LTCC organization, and the consequences to LTCC function and neuronal signaling. The project has three specific aims, which are to determine how selectively eliminating 1) Kv2.1 expression, 2) Kv2.1 clustering, and 3) the ability of Kv2.1 to enhance LTCC clustering impacts somatic LTCC localization and function, Ca2+-induced Ca2+ release or sparks, and LTCC-dependent transcript factor activation. The proposed studies have the potential of transforming our understanding of how neuronal ion channels are regulated and how this impacts Ca2+ signaling in health and when altered in disease.